Shake-flask culture of Laccaria laccata,an ectomycorrhizal basidiomycete

 Large-scale exploitation of the potential benefits of ectomycorrhizal fungi in improving plantation yields means that fermentation techniques for these fungi will be required. Starting with a base performance on a rich, complex medium, the effect of variations in some physicochemical culture parameters on biomass yield was studied. It was possible to reduce the amount of phosphate salts (to 1/9th) and other ingredients (to 1/3rd) in the medium. A shaking speed of either 100 rpm or 200 rpm in an orbital incubator was satisfactory and biomass yield responded to an increase in carbon substrate (glucose, from 10 g l^-1 and 20 g l^-1) though Y _x/s declined. An increase in inoculum size shortened culture time but decreased biomass yield. The upper limit of the incubation temperature was between 25°C and 30°C. Biomass yields were about 12 g l^-1 dry weight (Y _x/s=0.63) when 20 g l^-1 glucose was supplied, and about 7 g l^-1 (Y _x/s=0.74) when 10 g l^-1 glucose was supplied. Received: 9 October 1995/Accepted: 4 December 1995     

Transition rate kinetics from ethanol oxidation to glucose utilisation within a structured model of baker’s yeast

 The transition rate kinetics from ethanol oxidation to glucose utilisation, within a structured model of baker’s yeast, described previously, were experimentally identified. The shift in metabolism has been assessed through glucose pulses during batch growth on ethanol. The influence of glucose concentration (between 0.25 g l^-1 and 0.90 g l^-1) and initial biomass concentration (between 0.61 g l^-1 and 1.44 g l^-1) on the transition rate was determined. The transition rate can not be described by a first-order saturation-type kinetics with respect to glucose only. A corrective term, which takes into account biomass concentration should be included. Received: 28 April 1995/Received revision: 6 July 1995/Accepted: 22 August 1995     

Diatom composition and biomass variability in nearshore waters of Maxwell Bay, Antarctica, during the 1992/1993 austral summer

 Diatom composition and biomass were investigated in the nearshore water (<30 m in depth) of Maxwell Bay, Antarctica during the 1992/1993 austral summer. Epiphytic or epilithic diatoms such as Fragilaria striatula, Achnanthes brevipes var. angustata and Licmophora spp. dominated the water column microalgal populations. Within the bay, diatom biomass in surface water was several times higher at the nearshore (2.4–14 μg C l^-1) than at the offshore stations (>100 m) (1.2–3.2 μg C l^-1) with a dramatic decrease towards the bay mouth. Benthic forms accounted for >90% of diatom carbon in all nearshore stations, while in the offshore stations planktonic forms such as Thalassiosira antarctica predominated (50–>90%). Microscopic examination revealed that many of these diatoms have become detached from a variety of macroalgae growing in the intertidal and shallow subtidal bottoms. Epiphytic diatoms persistently dominated during a 19-day period in the water column at a fixed nearshore station, and the biomass of these diatoms fluctuated from 0.86 to 53 μg C l^-1. A positive correlation between diatom biomass and wind speed strongly suggests that wind-driven resuspension of benthic forms is the major mechanism increasing diatom biomass in the water column. Received: 28 April 1995/Accepted: 1 April 1996     

Octanoate increases cytosolic Ca2+ concentration and membrane conductance in ovine pancreatic acinar cells

 In order to investigate the cellular mechanisms involved in amylase release in response to stimulation with short-chain fatty acids, changes in intracellular calcium concentration ([Ca²⁺]_i), membrane current and amylase release were measured in pancreatic acinar cells of sheep. Both octanoate and acetylcholine raised [Ca²⁺]_i in acinar cells in a concentration-dependent manner. The rise in [Ca²⁺]_i in response to the stimulation with octanoate (10 mmol ⋅ l^-1) was reduced in a medium without CaCl₂, but was markedly enhanced by reintroduction of CaCl₂ into the medium up to 2.56 mmol ⋅ l^-1. Perfusion of the cells with a medium containing octanoate (5 mmol ⋅ l^-1) or acetylcholine (0.5 μmol ⋅ l^-1) immediately raised inward current across the cell membrane at a holding-membrane potential of −30 mV. The inward current became greater as the holding potential became more negative. The equilibrium potential was 1.8 mV and 3.9 mV for octanoate and acetylcholine, respectively, being consistent with that for Cl^-. Although intracellular application of octanoate through a patch-clamp pipette also raised inward current after several minutes in some cells (4 out of 12), this possibility was significantly smaller than that for extracellular application. In other cells, even though the intracellular application of octanoate did not cause an increase in current, it always caused responses immediately after introduction of the fatty acid into the medium. Stimulation with fatty acid as well as acetylcholine raised amylase release in a concentration-dependent manner in cells dispersed from tissue segments with crude collagenase and trypsin inhibitor. Without trypsin inhibitor, crude collagenase significantly and selectively reduced the octanoate (10 mmol ⋅ l^-1)-induced amylase release. Dispersion with crude collagenase and trypsin significantly reduced both responses induced by octanoate and acetylcholine (5.5 μmol ⋅ l^-1). We conclude that fatty acids and acetylcholine increase [Ca²⁺]_i, which consequently evokes a rise in transmembrane ion (Cl^-) conductance and amylase release, and that trypsin-sensitive protein(s) in the cell membrane are involved in secretory processes activated by stimulation with fatty acids in ovine pancreatic acinar cells. Accepted: 14 May 1996     

Effect of temperature and various nitrogen sources on L(+)-lactic acid production by Lactobacillus casei

 Two homofermentative strains, Lactobacillus casei NRRL B-441 and Lactobacillus casei subsp. rhamnosus NRRL B-445 were selected for further study from 17 lactic acid bacterial strains screened for lactic acid production. The effect of temperature on lactic acid production with the selected strains was investigated by adapting both strains to four different temperatures. The production of L(+)-lactic acid by both strains was most efficient at 37°C, although with L. casei the highest lactic acid concentration was obtained at 41°C. The maximal volumetric productivity with L. casei was 4.1 g l^-1 h^-1 and with L. casei subsp. rhamnosus 3.5 g l^-1 h^-1. The composition of the medium was studied in order to replace the costly yeast extract with less expensive sources of nitrogen and amino acids. From 11 different nitrogen sources investigated at 37°C, barley malt sprouts (88 g l^-1 lactic acid in 66 h) and grass extract (74 g l^-1 lactic acid in 73 h) were the best economic alternatives. The effect of different combinations of yeast extract, peptone and malt sprouts was further studied by using statistical experimental design, and an empirical second-order polynomial model was constructed on the basis of the results. With the right combination most of the yeast extract could be substituted by barley malt sprouts for efficient lactic acid production. A method for extraction of nutrients and growth factors from malt sprouts is also described. Received: 25 September 1995/Accepted: 24 October 1995     

Electrogenic cation transport across leech caecal epithelium

 Electrogenic cation transport across the caecal epithelium of the leech Hirudo medicinalis was investigated using modified Ussing chambers. Transepithelial resistance (R _T ) and potential difference (V _T ) were 61.0±3.5 Ω ⋅ cm² and −1.1±0.2 mV (n=149), respectively, indicating that leech caecal epithelium is a “leaky” epithelium. Under control conditions short circuit current (I _SC ) and transepithelial Na⁺ transport rate (I _Na ) averaged at 22.1±1.5 μA ⋅ cm^-2 and 49.7±2.6 μA ⋅ cm^-2, respectively. Mucosal application of amiloride (100 μmol ⋅ l^-1) or benzamil (50 μmol ⋅ l^-1) influenced neither I _SC nor I _Na . The transport system in the apical membrane showed no pronounced cation selectivity and a linear dependence on mucosal Na⁺ concentration. Removal of mucosal Ca²⁺ increased I _SC by about 50% due to an increase of transepithelial Na⁺ transport. Trivalent cations (La³⁺ and Tb³⁺, 1 mmol ⋅ l^-1 both) added to the mucosal Ringer solution reduced I _Na by more than 40%. Serosal ouabain (1 mmol ⋅ l^-1) almost halved I _SC and I _Na while 0.1% (=5.4 mmol ⋅ l^-1) DNP decreased I _Na to 11.8±5.1% of initial values. Serosal addition of cAMP increased both I _SC and I _Na whereas the neurotransmitters FMRFamide, acetylcholine, GABA, L-dopa, serotonin and dopamine failed to show any effects; octopamine, glycine and L-glutamate reduced I _Na markedly. On the basis of these results we conclude that in leech caecal epithelium apical uptake of monovalent cations is mediated by non-selective cation conductances which are sensitive to extracellular Ca²⁺ but insensitive to amiloride. Basolaterally Na⁺ is extruded via ouabain-sensitive and -insensitive ATPases. cAMP activates Na⁺ transport across leech caecal epithelium, although the physiological stimulus for cAMP-production remains unknown. Accepted: 20 May 1996     

Selection of an adhesive phenotype of Streptococcus salivarius subsp. thermophilus for use in fixed-bed reactors

 Streptococcus salivarius subsp. thermophilus was cultivated in a chemostat in order to obtain an adhesive phenotype of this strain. When the system was operated at low dilution rates (D<0.2 h^-1) for about 4 weeks, the strain formed a visible film on the surface of the culture vessel. The biofilm cells were not washed out even when dilution rates were increased (D=6.9 h^-1), and this resulted in a high biomass productivity (P=4.1 g l^-1h^-1). On the other hand, when the culture was grown at dilution rates faster than 0.2 h^-1, only the free suspended cells were present in the culture broth, and were washed out at velocities of about 1.0 h^-1. The biomass productivity was consequently lower (P=1.33 g l^-1h^-1) than in the previous case. The selected adhesive phenotype was grown on different glass beads and the possibility of lactate fermentation in a continuous and semicontinuous mode was demonstrated. Received: 16 August 1995/Received revision: 18 March 1996/Accepted: 25 March 1996     

Inhibition of Clostridium butyricum by 1,3-propanediol and diols during glycerol fermentation

1,3-Propanediol inhibition during glycerol fermentation to 1,3-propanediol by Clostridium butyricum CNCM 1211 has been studied. The initial concentration of the 1,3-propanediol affected the growth of the bacterium more than the glycerol fermentation. μ _max was inversely proportional to the initial concentration of 1,3-propanediol (0–65 g l⁻¹). For glycerol at 20 g l⁻¹, the growth and fermentation were completely stopped at an initial 1,3-propanediol concentration of 65 g l⁻¹. However, for an initial 1,3-propanediol concentration of 50 g l⁻¹ and glycerol at 70 g l⁻¹, the final concentration (initial and produced) of 1,3-propanediol reached 83.7 g l⁻¹(1.1 M), with complete consumption of the glycerol. Therefore, during the fermentation, the strain tolerated a 1,3-propanediol concentration higher than the initial inhibitory concentration (65 g l⁻¹). The addition of 1,2-propanediol or 2,3-butanediol (50 g l⁻¹) in the presence of glycerol (50–100 g l⁻¹), showed that 2-diols reduced the μ _max in a similar way to 1,3-propanediol. The measurement of the osmotic pressure of glycerol solutions, diols and diol/glycerol mixtures did not indicate any differences between these compounds. The hypothesis of diol inhibition was discussed. Taking into account the strain tolerance of highly concentrated 1,3-propanediol during fermentation, the fermentation processes for optimising production were considered. Received: 15 November 1999 / Revision received: 1 February 2000 / Accepted: 4 February 2000     

Cardiac responses to salinity variations in two differently zoned Mediterranean limpets

Cardiac activity of two Mediterranean limpets was tested at different salinities. Patella caerulea inhabits the lower midlittoral where it is exposed to variations in salinity, while P. aspera experiences more stable salinity conditions in the infralittoral fringe. When exposed to moderate hypo- and hypersalinity (23 g l⁻¹ and 43 g l⁻¹) for 24 min, P. caerulea showed no significant variation in heart rate with respect to the control salinity (33 g l⁻¹), while P. aspera exhibited a significant increase in heart rate in both conditions. This suggests a rise in metabolic rate due to activation of behavioural responses or physiological regulation. When exposed to extremely low salinity (3 g l⁻¹) for 24 min, heart contractions ceased in most specimens of P. caerulea. A smaller number of specimens also displayed cessation of heart beat when exposed to extremely high salinity (63 g l⁻¹). The heart beat resumed quickly in all specimens when they were returned to control salinity conditions. In contrast, cardiac activity was not interrupted in any of the P. aspera specimens at the 3 g l⁻¹ and 63 g l⁻¹ salinity levels, but strong bradycardia was evident. Contractile activity of the heart ceased in all specimens of P. caerulea and P. aspera when they were exposed to prolonged hypo-osmotic stress (3 g l⁻¹ for 24 h). This acardia was largely reversible in P. caerulea, but most specimens of P. aspera did not recover from the treatment. Accepted: 3 July 1999     

Continuous production of l(+)-lactic acid by Lactobacillus casei in two-stage systems

A two-stage two-stream chemostat system and a two-stage two-stream immobilized upflow packed-bed reactor system were used for the study of lactic acid production by Lactobacillus casei subsp casei. A mixing ratio of D ₁₂/D ₂= 0.5 (D = dilution rate) resulted in optimum production, making it possible to generate continuously a broth with high lactic acid concentration (48 g l⁻¹) and with a lowered overall content of initial yeast extract (5  g l⁻¹), half the concentration supplied in the one-step process. In the two-stage chemostat system, with the first stage at pH 5.5 and 37 °C and a second stage at pH 6.0, a temperature change from 40 °C to 45 °C in the second stage resulted in a 100% substrate consumption at an overall dilution rate of 0.05 h⁻¹. To increase the cell mass in the system, an adhesive strain of L. casei was used to inoculate two packed-bed reactors, which operated with two mixed feedstock streams at the optimal conditions found above. Lactic acid fermentation started after a lag period of cell growth over foam glass particles. No significant amount of free cells, compared with those adhering to the glass foam, was observed during continuous lactic acid production. The extreme values, 57.5 g l⁻¹ for lactic acid concentration and 9.72 g l⁻¹ h⁻¹ for the volumetric productivity, in upflow packed-bed reactors were higher than those obtained for free cells (48 g l⁻¹  and 2.42 g l⁻¹ h⁻¹) respectively and the highest overall l(+)-lactic acid purity (96.8%) was obtained in the two-chemostat system as compared with the immobilized-cell reactors (93%). Received: 4 December 1997 / Received revision: 23 February 1998 / Accepted: 14 March 1998     

Pentachlorophenol biodegradation kinetics of an oligotrophic fluidized-bed enrichment culture

A fluidized-bed reactor (FBR) was used to enrich an aerobic chlorophenol-degrading microbial culture. Long-term continuous-flow operation with low effluent concentrations selected oligotrophic microorganisms producing good-quality effluent for pentachlorophenol(PCP)-contaminated water. PCP biodegradation kinetics was studied using this FBR enrichment culture. The results from FBR batch experiments were modeled using a modified Haldane equation, which resulted in the following kinetic constants: q _max = 0.41 mg PCP mg protein⁻¹ day⁻¹, K _S = 16 μg l⁻¹, K _i = 5.3 mg l⁻¹, and n = 3.5. These results show that the culture has a high affinity for PCP but is also inhibited by relatively low PCP concentrations (above 1.1 mg PCP l⁻¹). This enrichment culture was maintained over 1 year of continuous-flow operation with PCP as the sole source of carbon and energy. During continuous-flow operation, effluent concentrations below 2 μg l⁻¹ were achieved at 268 min hydraulic retention time (t _HR) and 2.5 mg PCP l⁻¹ feed concentration. An increase in loading rate by decreasing t _HR did not significantly deteriorate the effluent quality until a t _HR decrease from 30 min to 21 min resulted in process failure. Recovery from process failure was slow. Decreasing the feed PCP concentration and increasing t _HR resulted in an improved process recovery. Received: 10 October 1996 / Received revision: 21 January 1997 / Accepted: 24 January 1997     

Microbial sensors for naphthalene using Sphingomonas sp. B1 or Pseudomonas fluorescens WW4

 Amperometric biosensors for naphthalene were developed using either immobilized Sphingomonas sp. B1 or Pseudomonas fluorescens WW4 cells. The microorganisms were immobilized within a polyurethane-based hydrogel, which was used for a microbial biosensor for the first time. Both strains were shown to be equally suited for the quantification of naphthalene in aqueous solutions. The biosensors were tested in a flow-through system and a stirred cell (batch method). In both systems a linear response down to the detection limit was obtained. Measurements in the flow-through system gave sensitivities of up to 1.2 nA mg⁻¹ l⁻¹ and a linear range from 0.03 mg/l to 2.0 mg/l. The response time (t ₉₅) was 2 min and the sample throughput six per hour; the repeatability was within ±5 %. With the batch method, sensitivities of between 3 nA mg⁻¹ l⁻¹ and 5 nA mg⁻¹l⁻¹ and a linear range of 0.01–3.0 mg/l were obtained; the response time was between 3 min and 5 min. The sensors reached an operational lifetime of up to 20 days. The sensitivity of both sensors for naphthalene was, in most cases, more than four times higher than for various other substrates. Received: 18 October 1995/Received revision: 22 December 1995/Accepted: 22 January 1996     

The effect of inoculation and the type of carrier material used on the biofiltration of methyl sulphides

 Low elimination capacities (less than 10 g m^-3 day^-1) were observed for the odorant dimethyl sulphide (Me₂S) when either wood bark or compost was used as the carrier material in a laboratory-scale biofilter. Enrichment experiments were set up by incubation of garden soil samples during 4 weeks with 100 ppm (v/v) headspace concentrations of both Me₂S and dimethyl disulphide (Me₂S₂). After transfer to a mineral medium, Me₂S- and Me₂S₂-degrading enrichment cultures were obtained for all five soil samples tested, both compounds being converted stoichiometrically to sulphuric acid. Upon inoculation of the laboratory-scale biofilter with one of these enrichment cultures (±120 g cell dry weight m^-3 reactor), the elimination capacity for Me₂S increased in a 3-week period to 35 g m^-3 day^-1 and 680 g m^-3 day^-1 when wood bark and compost were used as the respective carrier materials. Both inoculated biofilters were able to degrade Me₂S₂, however the elimination capacities obtained for Me₂S₂ were lower (e.g. 24 g m^-3 day^-1 for the wood bark filter) compared to those for Me₂S. For both inoculated biofilters, a gradual decrease of the elimination capacity for the methyl sulphides was observed as a result of acidification of the carrier material, suggesting that pH regulation is necessary if long-term biofiltration experiments are to be performed. Received: 6 June 1995/Received revision: 10 August 1995/Accepted: 22 August 1995     

Continuous production of ethanol using yeast cells immobilized in preformed cellulose beads

 Saccharomyces cerevisiae cells were immobilized on preformed cellulose beads by adsorption. The fermentation capacity of the immobilized yeast cells was found to be practically independent of the hydrogen ion concentration between pH 3.1 and 6.25. The fermentation capacity was maximal at 30 °C. The immobilized yeast cells were used for continuous production of ethanol in a fluidized-bead reactor. The average values characteristic for the process were an ethanol concentration of 41.9±0.1 g l^-1, a fermentation efficiency of 82.9±2.1% and a volumetric productivity of 3.94±0.52 g l^-1 h^-1. Received: 9 October 1995/Accepted: 22 April 1996     

Effects of saturated fatty acids on amylase release from exocrine pancreatic segments of sheep,rats, hamsters,field voles and mice

 Stimulatory effects of saturated fatty acids consisting of 4 (butyrate), 8 (octanoate), 12 (laurate) and 16 (palmitate) carbon atoms, as well as acetylcholine on pancreatic amylase release were assessed in tissue segments isolated from sheep, rats, hamsters, field voles and mice. The amount of amylase release induced by the fatty acids (1 μmol ⋅ l^-1 to 10 mml ⋅ l^-1) and by acetylcholine (10 nmol ⋅ l^-1 to 100 μmol ⋅ l^-1) increased in a concentration-dependent manner, and the maximum response in response to the fatty acids was obtained at the maximal dose used. The maximum increase in amylase release in response to butyrate or octanoate was highly and significantly (r=0.974, P<0.001) dependent on the log value of the mean body mass in the following order: sheep>rats>hamsters>field voles>mice. On the other hand, the response to laurate and palmitate was variable among animal species. Addition of atropine (1.4 μmol ⋅ l^-1) to the medium did not reduce the responses to octanoate stimulation, but significantly reduced acetylcholineinduced responses, implying that the effects of the fatty acids were not mediated through activation of muscarinic acetylcholine receptors. Reduction of calcium ion concentration in the medium significantly inhibited the responses induced by the fatty acids and acetylcholine, suggesting that amylase release depends on extracellular calcium ions. Accepted: 14 May 1996     

Candida bombicola : production of novel alkyl glycosides based on glucose/2-dodecanol

Candida bombicola produces glycolipids containing sophorose and a glycosidically/esterically bound ω- or (ω−1)-hydroxy C₁₆₍₁₈₎ acid. Here we describe novel glycolipids from this source. Glucose and 2-dodecanol were used for the cultivation of the yeast, one part of the racemic secondary alcohol being connected directly with a glucose or a sophorose unit. A relatively high content of yeast extract, up to 4 g l⁻¹, and subsequently higher biomass concentrations favoured the production of novel products. The provision of 150 g l⁻¹ glucose and 15 g l⁻¹ 2-dodecanol resulted in maximum production of 22 g l⁻¹ novel alkyl glycosides (more than 90% novel products). The molecular structures were analysed by gas chromatography, fast atom bombardment/mass spectrometry, ¹H- and ¹³C-nuclear magnetic resonance and optical rotation studies. Sophorose and glucose were detected as carbohydrate moieties, (S)-(+)-2-dodecanol (88%) was found to be the major lipid moiety. The new glycolipids are suitable biosurfactants, reducing the surface tension of water from 72 mN m⁻¹ to 32–38 mN m⁻¹. Received: 8 December 1997 / Received revision: 19 March 1998 / Accepted: 20 March 1998     

Isolation of <Emphasis Type="Italic">Aspergillus oryzae</Emphasis> mutants for heterologous protein production from a double proteinase gene disruptant

The recombinant Pichia pastoris harboring an improved methionine adenosyltransferase (MAT) shuffled gene was employed to biosynthesize S-adenosyl-l-methionine (SAM). Two l-methionine (l-Met) addition strategies were used to supply the precursor: the batch addition strategy (l-Met was added separately at three time points) and the continuous feeding strategies (l-Met was fed continuously at the rate of 0.1, 0.2, and 0.5 g l⁻¹ h⁻¹, respectively). SAM accumulation, l-Met conversion rate, and SAM productivity with the continuous feeding strategies were all improved over the batch addition strategy, which reached 8.46 ± 0.31 g l⁻¹, 41.7 ± 1.4%, and 0.18 ± 0.01 g l⁻¹ h⁻¹ with the best continuous feeding strategy (0.2 g l⁻¹ h⁻¹), respectively. The bottleneck for SAM production with the low l-Met feeding rate (0.1 g L⁻¹ h⁻¹) was the insufficient l-Met supply. The analysis of the key enzyme activities indicated that the tricarboxylic acid cycle and glycolytic pathway were reduced with the increasing l-Met feeding rate, which decreased the adenosine triphosphate (ATP) synthesis. The MAT activity also decreased as the l-Met feeding rate rose. The reduced ATP synthesis and MAT activity were probably the reason for the low SAM accumulation when the l-Met feeding rate reached 0.5 g l⁻¹ h⁻¹.     

Osmotic stress effects on the freezing tolerance of the antarctic nematodePanagrolaimus davidi

 The freezing and freezing survival of the Antarctic nematode Panagrolaimus davidi after exposure to solutions of different osmotic concentrations has been examined using a thermoelectric cooling stage and multi-specimen cooling block to see if there is any evidence that freeze-induced desiccation prevents inoculative freezing. The nematodes froze in all the test solutions used (up to 1138 mosmol ⋅ l^-1) and at all cooling rates and nucleation temperatures tested. Freezing survival was at its maximum in 0.1 mol ⋅ l^-1 NaCl in artificial tap water after 1 h exposure to the test solution and in artificial tap water after 24 h exposure. Hyperosmotic and hyposmotic stress adversely affected the nematodes’ ability to survive freezing. In non-frozen controls survival declined with increasing osmolality of the test solution. Measurements of the osmolality of water extracted from a variety of moss samples indicate that the nematodes are exposed to an osmotic concentration of about 9 mosmol ⋅ l^-1 in their natural habitat. This is close to that of artificial tap water. Our experiments, and measurements of freeze concentration effects in the literature, indicate that freeze-induced desiccation is unlikely to prevent inoculative freezing and the survival of nematodes over the winter. Accepted: 5 May 1996     

Physiology of acute silver toxicity in the starry flounder (Platichthys stellatus) in seawater

Physiological effects of exposure to silver (AgCl_n ⁿ⁻¹; 250 μg Ag l⁻¹ or 1000 μg Ag l⁻¹) in seawater fish were investigated using adult starry flounders. While all fish survived up to 10 days in 250 μg Ag l⁻¹, flounders started to die after day 4 in 1000 μg l⁻¹. Dose-dependent increases in plasma and hepatic silver concentrations showed that silver was available for uptake. There were minimal negative effects on hematological parameters, acid-base status, and blood gases. Plasma ammonia showed a pronounced (three- to four-fold), but transient increase in flounders exposed to either 250 μg Ag l⁻¹ or 1000 μg Ag l⁻¹. Whole body ammonia and acid equivalent efflux measurements indicated that ammonia retention was due to a combination of stimulated production and inhibited excretion. In the 1000-μg Ag l⁻¹ group there was a similar transient increase in plasma [magnesium], which was restored by day 4. In contrast, plasma chloride and sodium levels increased gradually towards the point when fish began to die. At 250 μg Ag l⁻¹, the Na⁺/K⁺-ATPase activity of the intestine was unaffected but there was a two-fold increase in branchial Na⁺/K⁺-ATPase activity. The latter effect was interpreted as compensation for an elevated chloride and sodium load. The increases in plasma chloride and sodium concentrations were accompanied by a marked suppression of drinking, thereby indicating that acute silver toxicity was likely caused by a combination of elevated electrolyte concentrations and dehydration. Accepted: 9 June 1999     

Production of polyhydroxybutyrate by Ralstonia eutropha from protein hydrolysates

Polyhydroxybutyrate (PHB) was produced by Ralstonia eutropha DSM 11348 (formerly Alicaligenes eutrophus) in media containing 20–30 g l⁻¹ casein peptone or casamino acids as sole sources of nitrogen. In fermentations using media based on casein peptone, permanent growth up to a cell dry mass of 65 g l⁻¹ was observed. PHB accumulated in cells up to 60%–80% of dry weight. The lowest yields were found in media without any trace elements or with casamino acids added only. The residual cell dry masses were limited to 10–15 g l⁻¹ and did not contain PHB. The highest productivity amounted to 1.2 g PHB l⁻¹ h⁻¹. The mean molecular mass of the biopolymer was determined as 750 kDa. The proportion of polyhydroxyvalerate was less than 0.2% in PHB. The bioprocess was scaled up to a 300-l plant. During a fermentation time of 39 h the cells accumulated PHB to 78% w/w. The productivity was 0.98 g PHB l⁻¹ h¹. Received: 8 July 1998 / Accepted: 26 August 1998